Many documents describe the measurement of various different analytes in chronic and acute wound fluid. The following documents describe the measurement of endogenous protease enzymes in wound fluid.
C. N. Rao et al. in the Journal of Investigative Dermatology, vol. 105(4), pages 572-578 (1995) describe the results of analysing chronic and acute wound fluids for elastase, alpha-1-antitrypsin (AAT) and fibronectin. It was found that the elastase level was 10 to 40 times higher in the chronic wound fluid.
US-A-2003/0119073 describes sensors for the assay of catabolic protease enzymes in wound fluid. The analyte enzymes include human neutrophil elastase (hNE). It is suggested therein that the invention can be used in a method of treating chronic wounds by detecting the presence of catabolic protease enzymes, and then treating the wound with inhibitors that are specific for the detected enzymes.
WO 2006/030232 describes a diagnostic test apparatus for determining a ratio of: (a) at least one endogenous protease enzyme inhibitor, to (b) at least one endogenous protease enzyme, in a sample of a wound fluid. The apparatus is used in a method of treating a wound, and used for identifying wounds that are likely to respond well to treatment with oxidized regenerated cellulose (ORC) containing dressings. For example, the apparatus is used for identifying prospective responders and non-responders to treatment of wounds with PROMOGRAN™ collagen/ORC wound dressing.
John F. Tarlton et al. in Wound Repair and Regeneration, Vol. 7(5), pages 347-355 (1999) describe an academic study into the prognostic value of markers of collagen remodelling in venous ulcers. The following markers were studied: proMMP-2, proMMP-9, neutrophil elastase (hNE), MMP-2 and MMP-9. The data showed that expression of MMPs-2 and 9 and hNE were higher in venous ulcer exudates than in acute wound fluids. Also studied was a comparison of the above markers in healing versus non-healing ulcers. A further study was performed in which wound fluids were compared from healing and non-healing regions of the same ulcer in order to eliminate patient-to-patient variation. The data showed only very weak correlation, if any, of MMP-2 and MMP-9 to wound healing status in the multiple-patient study. The correlation for neutrophil elastase was also quite weak. However, the comparison experiments done on wound fluids from different regions of the same wound did show a statistically significant correlation of neutrophil elastase to wound healing status, and a weakly statistically significant correlation of MMP-9 to wound healing status.
Alison G. Patrick et al. in Macromolecular Bioscience, Vol. 10, pages 1184-1193 (2010) describe hydrogel particles having surface bonded moieties for the detection of elastase, MMP-1 and/or MMP-12. The moieties consist of an enzyme-specific peptide substrate linking two FRET chromophores, whereby cleavage of the peptide results in a fluorescence signal. The peptides are selective for elastase, or for MMP-1 and MMP-12. It is mentioned that MMPs are associated with the progression of chronic wounds.
Ralf Smeets et al. in International Wound Journal, Vol. 5(2), pages 195-203 (2008) describe the effect of collagen/ORC sponge dressing treatment on “gelatinase activity”, and also on levels of MMP-2, and elastase in chronic wounds. The results show that collagen/ORC does not significantly effect the MMP-2 level, but that it significantly reduces the gelatinase and elastase activities.
Cornelia Wiegand et al. in Archives of Dermatological Research, Vol. 302, pages 419-428 (2010) describe measurement of various proteases in acute chronic wound fluids. The measured proteases were neutrophil elastase, MMP-2 and MMP-13. These were all found to be elevated in chronic wounds relative to acute wounds. Levels of these proteases were found to decrease in the course of wound healing.
Breda Cullen et al. in Wound Repair and Regeneration, Vol. 15 (6) page A148 (2007) describe a comparison of collagen/ORC/silver dressings with other collagen (+silver) dressings in the treatment of chronic wounds. The results demonstrate that while the collagen containing dressings could remove MMP-type proteases, not all inactivate elastase. However, the collagen/ORC/silver dressings significantly reduced the levels of both elastase and total MMP.
WO-A-03058237 describes sensors for detection of various protease enzymes in wound fluid. The analytes include MMP-1, MMP-8. MMP-9 and neutrophil elastase, either singly or simultaneously. The specification also suggests then treating the wound with inhibitors that are specific for protease enzymes found in the wound. The sensors described in the specification are all immunological.
GB-A-2422664 refers to measurement of various analytes, including MMP-1, MMP-8, MMP-9 and neutrophil elastase in wound fluid. Peptide sequences are disclosed that are cleavable by elastase, MMP-2 or MMP-9. Also disclosed are devices with multiple lateral flow paths for measuring multiple analytes.
WO-A-2008070865 describes methods for evaluating the status of the healing process of a wound comprising contacting wound fluid with a cleavable peptide. The method may be used to detect at least one protease selected from the group of MMP-2, MMP-8, MMP-9 and elastase.
WO98/00180 and EP-A-1153622 describe the use of freeze-dried sponges comprising oxidized regenerated cellulose (ORC), optionally admixed with collagen, for the treatment of chronic wounds. Dressings based on oxidized cellulose have been found to give outstanding results in the treatment of chronic wounds, including diabetic ulcers, venous ulcers and decubitis ulcers.
WO 2004/024197 and EP-A-1536845 describe the use of wound dressing materials comprising complexes of anionic polysaccharides with silver. In particular, wound dressing materials comprising complexes formed between anionic polysaccharides, such as ORC, and silver, and to the uses thereof for the treatment of wounds are disclosed.
GB-A-2393120 describes the use of wound dressings based on ORC in combination with chitosan for the treatment of chronic wounds. The dressings are shown to reduce the levels of elastase and collagenase in the wound fluids.
It will be apparent from the foregoing that levels of matrix metalloproteinases and human neutrophil elastase levels are thought to be elevated in chronic wound fluid relative to acute wound fluid. Changes in the levels of these markers may be correlated to wound healing. However, the above references do not show how to use these markers to distinguish between healing and non-healing chronic wounds. For example, Tarlton et al. found little or no statistically significant correlation between these markers and healing/non-healing chronic wound type in inter-patient studies. Furthermore, protease levels in any sample of wound fluid appear to be poorly correlated. Thus, a wound having high levels of hNE may not also have elevated levels of MMP. This may be due to interactions between proteases. For example hNE acts as a physiological activator of MMPs including MMP-9 which is synthesised in a latent, inactive form. Additionally, there is some biochemical redundancy between the proteases; although some proteases are more efficient than others there is a crossover and synergy of substrates between these proteases, for example elastin can be degraded by both MMP-9 and Elastase, and both MMP-8 and MMP-9 are required to digest Collagen type I. Therefore it may be possible for a non-healing wound to be low in one protease but this could be compensated by an excess of another protease.
Thus, it is an object of the present invention to provide improved means to distinguish healing from non-healing chronic wounds. It is a further object to provide such a means that can be performed on a sample of wound fluid removed from the wound. It is a further object to provide a method that requires only a measurement made at a single point in time, in contrast to the measurement of wound area, which requires multiple measurements over an extended period. It is a further object of the invention to avoid unnecessary special therapy on other patients who present with healing wounds. Preferably, the present invention seeks to identify a sub-group of patients wherein the probability of their wound being non-healing is at least about 80%, suitably at least about 90%.
It has also been found that a sub-group of chronic wound patients exhibit a particularly large improvement in wound healing when treated with collagen/ORC sponges.
Thus, it is also an object of the present invention to provide alternative or improved means to identify as early as possible the sub-group of patients that exhibit a particularly large improvement in wound healing when treated with oxidized cellulose so that they can receive maximum benefit from this therapy. It is a further object of the invention to avoid unnecessary oxidized cellulose therapy on other patients who may be less likely to benefit. Accordingly, it is an object of the present invention to identify which patients would be more likely to respond to treatment with oxidised cellulose before treatment with oxidised cellulose has begun